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Studies of the structure and function of recombinant human hephaestin Vashchenko, Ganna


Hephaestin is a multicopper ferroxidase involved in iron absorption in the small intestine. The ferroxidase activity of hephaestin is thought to play an important role during iron export from intestinal enterocytes and the subsequent iron loading of the blood protein transferrin, which delivers iron to the tissues. Structurally, the ectodomain of hephaestin is predicted to resemble ceruloplasmin, the soluble ferroxidase of blood. In this work I investigated substrate specificity, copper loading and the ferroxidation mechanism of recombinantly expressed human hephaestin. The hephaestin ectodomain (Fet3Hp) was expressed in Pichia pastoris and purified to electrophoretic homogeneity by immunoaffinity chromatography. Recombinant hephaestin retained ferroxidase activity and showed an average copper content of 4.2 copper atoms per molecule. The Km values of Fet3Hp for such organic substrates as p-phenylenediamine and o-dianisidine were close to values determined for ceruloplasmin. However, in contrast to ceruloplasmin, recombinant hephaestin was incapable of direct oxidation of adrenaline and dopamine implying a difference in biological substrate specificities between these two homologous oxidases. I also expressed hephaestin ectodomain with the ceruloplasmin signal peptide (CpHp) using BHK cells as an expression system. Ion exchange chromatography of purified CpHp resulted in the production of a hephaestin fraction with improved catalytic and spectroscopic properties. Detailed kinetic analysis of CpHp ferroxidation rates revealed the presence of two types of iron-binding sites with different affinities towards ferrous iron. Michaelis constants for high- and low-affinity ferrous binding sites in CpHp were comparabale to the corresponding Km values in ceruloplasmin suggesting that both paralogs utilize similar amino acid residues for iron binding. To investigate the role of particular residues in iron specificity of hephaestin, mutations of putative iron ligands were introduced into CpHp using site-directed mutagenesis. Kinetic analysis of ferroxidation rates of wild-type CpHp and variants revealed the important roles of residues E960 and H965 in hephaestin ferroxidase activity.

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